V High-dosevitaminD reducesdeficiencycausedbylowUVBexposureandlimitsHIV-1replicationinurbanSouthernAfricans

High-dose vitamin D ₃ reduces deficiency caused by low UVB exposure and limits HIV-1 replication

in urban Southern Africans

Anna K. Coussens

, Celeste E. Naude

, Rene Goliath

, George Chaplin

, Robert J. Wilkinson

, and Nina G. Jablonski

a

Clinical Infectious Diseases Research Initiative, Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Observatory 7925, South Africa;

Centre for Evidence-based Health Care, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg 7505, South Africa;

c

Department of Geography, The Pennsylvania State University, University Park, PA 16802;

Department of Anthropology, The Pennsylvania State University, University Park, PA 16802;

Stellenbosch Institute for Advanced Studies, Stellenbosch 7600, South Africa;

The Francis Crick Institute, Mill Hill Laboratory, London NW7 1AA, United Kingdom; and

Department of Medicine, Imperial College London, London W2 1PG, United Kingdom

Edited by Barry R. Bloom, Harvard School of Public Health, Boston, MA, and approved May 19, 2015 (received for review January 17, 2015)

Cape Town, South Africa, has a seasonal pattern of UVB radiation and a predominantly dark-skinned urban population who suffer high HIV-1 prevalence. This coexistent environmental and pheno- typic scenario puts residents at risk for vitamin D deficiency, which may potentiate HIV-1 disease progression. We conducted a longitudinal study in two ethnically distinct groups of healthy young adults in Cape Town, supplemented with vitamin D

in winter, to determine whether vitamin D status modifies the re- sponse to HIV-1 infection and to identify the major determinants of vitamin D status (UVB exposure, diet, pigmentation, and genet- ics). Vitamin D deficiency was observed in the majority of subjects in winter and in a proportion of individuals in summer, was highly correlated with UVB exposure, and was associated with greater HIV-1 replication in peripheral blood cells. High-dosage oral vita- min D

supplementation attenuated HIV-1 replication, increased circulating leukocytes, and reversed winter-associated anemia. Vi- tamin D

therefore presents as a low-cost supplementation to im- prove HIV-associated immunity.

seasonal variation | infectious disease | polymorphism | pigmentation |

nutrition

V itamin D is recognized as having diverse physiological and immunomodulatory functions, and deficiency is associated with a range of communicable and noncommunicable diseases, including HIV/AIDS progression and mortality (1, 2). Pre- vitamin D

is made in skin when UVB photons react with 7-dehydrocholesterol (7-DHC) in the cell membranes of kerati- nocytes (3). Constitutive and facultative skin pigmentation reg- ulates the penetration of UV radiation into the skin, because eumelanin competes with 7-DHC for UVB photons, thus con- trolling the availability of UVB for previtamin D

production (4).

Therefore people with high eumelanin content (and thus darker skin) require greater UVB exposure to make previtamin D

and are more prone to vitamin D deficiency. This requirement for greater exposure is exacerbated by seasonal UVB fluctua- tion; generally UVB levels exhibit a winter decline at latitudes

>30° (5, 6).

Serum vitamin D [25-hydroxy-vitamin D, 25(OH)D] levels are determined by skin (dermal and epidermal) production, dietary intake, storage, and turnover. Each of these determinants is modified at a variety of levels: skin production by pigmentation, age, and the time, duration, and level of UVB exposure; dietary intake by vitamin D composition of foods; storage by body mass index (BMI) (7) and serum vitamin D-binding protein (DBP) (8); and turnover by polymorphic variation in genes encoding metabolizing enzymes [cytochrome P450 (CYP) 2R1, CYP27B1, CYP24A1, 7-DHC reductase (DHCR7)], the vitamin D receptor (VDR) and DBP (9–12), age (13), infection (14, 15), serum calcium and parathyroid hormone concentrations (16), and smok- ing habit (17). The two most significant determinants are UVB

exposure and dietary intake, and although deconvolution of their relative impact is vital for understanding how to maintain vita- min D sufficiency for disease prevention, they are rarely inves- tigated in the same study, nor are results adjusted for all other confounders. Furthermore, the majority of studies investigate vitamin D status in relation to chronic disease; there is a dearth of information regarding the determinants of vitamin D sufficiency in the healthy state and its relation to disease prevention.

Cape Town has a diverse population with respect to ethnicity, skin pigmentation, and socioeconomic status. The Xhosa, who migrated as part of the geographic expansion of north and northwest African agriculturalists, are a significant proportion of the population. Another major population group comprises people of self-identified Cape Mixed ancestry, who represent a complex admixture of Xhosa, Khoisan (the oldest inhabitants), European, South Asian, and Indonesian populations (18). Given their exposure to seasonal UVB variation and high infectious disease risk, the peoples of the Cape are of particular importance in studying the determinants and immunological consequence of vitamin D status. In the context of greatest HIV-1 risk, those of particular importance are females aged 15–24 y and males aged 20–29 y (19).

We thus undertook a longitudinal study of healthy young adults in Cape Town to assess the relative contribution of pigmentation, seasonal UVB exposure, dietary vitamin D intake,

Significance

Vitamin D deficiency is associated with HIV/AIDS progression and mortality. Seasonal decline in UVB radiation, darkly pig- mented skin, low nutritional vitamin D intake, and genetic variation can increase risk of deficiency. Cape Town, South Africa, has a seasonal UVB regime and one of the world ’s highest rates of HIV-1 infection, peaking in young adults. In two ethnically distinct groups of young adults in Cape Town we found high prevalence of seasonal vitamin D deficiency resulting from inadequate UVB exposure. This deficiency was associated with increased permissiveness of blood cells to HIV- 1 infection which was reversed by vitamin D

supplementation.

Vitamin D may be a simple, cost-effective intervention, partic- ularly in resource-poor settings, to reduce HIV-1 risk and disease progression.

Author contributions: A.K.C., C.E.N., R.G., G.C., R.J.W., and N.G.J. designed and in- terpreted research; A.K.C., C.E.N., and R.G. performed research; A.K.C., C.E.N., G.C., and N.G.J. analyzed data; and A.K.C., C.E.N., G.C., and N.G.J. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.

1To whom correspondence should be addressed. email: [email protected].

This article contains supporting information online atwww.pnas.org/lookup/suppl/doi:10.

1073/pnas.1500909112/-/DCSupplemental.

MEDICALSCIENCESEARTH,ATMOSPHERIC, ANDPLANETARYSCIENCES

genetic variation, serum DBP, and smoking habit on vitamin D status and investigated whether seasonal vitamin D variation and vitamin D supplementation impact HIV-1 immunity in this high- risk population.

Results

Highly Prevalent Seasonal Vitamin D Deficiency and Its Reversal by Winter Supplementation. One hundred healthy (asymptomatic, BMI <30) young (18- to 24-y-old) adults (Xhosa, n = 50; Cape Mixed, n = 50) were recruited from two neighboring districts of Cape Town in the summer and were reassessed in the winter (for loss to follow-up, see Fig. S1). In winter, all participants received cholecalciferol (50,000 IU) weekly for 6 wk, and 30 Xhosa par- ticipants were followed up for 6 wk after their winter visit (Fig.

1A). All enrolled participants were asymptomatic with no evi- dence of infection (Fig. S2 A–C). The populations were well matched with similar female:male ratios and only a small dif- ference in age (Xhosa 21 y vs. Cape Mixed 18.5 y; P < 0.0001) and smoking status (Xhosa 50% vs. 18%; P = 0.0014) (Table S1).

Xhosa participants had darker skin pigmentation as measured by upper inner arm and forehead melanin index (MI) and erythema index (EI, a measure of tanning) (P < 0.0001; Table S2).

Although their higher melanin content reduced the rate of skin vitamin D production, Xhosa participants actually had higher serum 25(OH)D levels in summer than Cape Mixed participants (median 72.6 vs. 65.5 nmol/L; P = 0.038, Table 1).

Cape Mixed participants also had a trend toward greater vitamin D deficiency (<50 nmol/L) in summer (16 vs. 4%; P = 0.077).

Conversely, there was no difference in 25(OH)D levels between population groups in winter, when a significant drop in 25(OH)D levels was observed in both populations (P < 0.0001) and the majority of participants became vitamin D deficient (Fig. 1B).

Severe deficiency (<30 nmol/L) occurred in 18% of Xhosa par- ticipants and 12% of Cape Mixed participants in the winter, and overall 64% and 70%, respectively, had deficient serum 25(OH)D levels (Table 1). After winter supplementation, 77% of the Xhosa group gained optimal levels (≥75 nmol/L) [median126.4 nmol/L, interquartile range (IQR) 74.631–57.1 nmol/L] (Fig. 1C), and there was no change in corrected serum calcium (winter mean ± SD 2.31 ± 0.08 mmol/L vs. postsupplementation 2.34 ± 0.09 mmol/L) (Fig. S2D). Cape Mixed females had lower 25(OH)D levels in winter than Cape Mixed males (median 41.46 vs. 50.80 nmol/L;

P = 0.0054), and Xhosa females had lower 25(OH)D levels after

supplementation than Xhosa males (median 113.3 vs. 147.9 nmol/L, P = 0.047), indicating that females in both groups are at risk for lower 25(OH)D (Fig. 1 D and E).

Personal UVB Exposure, but Not Diet, Varies by Season. Dietary in- take of vitamin D was estimated using a 7-d quantitative food frequency questionnaire administered at each study visit. According to the estimated average requirement (EAR) cutoff-point method, 78–88% of participants in both populations had intakes below the EAR (400 IU) throughout the year, with median intakes ranging from 170 to 235 IU across groups and seasons (Fig. 1F and Table 1).

There was no difference in intake between sexes in the Xhosa participants, but Cape Mixed females had lower intakes than Cape Mixed males in both seasons (P < 0.001), mirroring the sex patterns observed for 25(OH)D levels (Fig. 1 E, G, and H).

To understand the extent to which UVB exposure contributes to vitamin D deficiency, solar UVB was monitored daily for the duration of the study, and participants completed sun-exposure questionnaires. Both groups spent significantly longer in the sun in summer than in winter (P ≤ 0.014), with personal net UVB (PNUVB) exposure more than 10-fold higher in summer (P <

0.0001, Fig. 1I). Xhosa participants spent ∼4 h longer each week in the sun in both seasons than Cape Mixed participants (me- dian: 1,335 vs. 1,096 minutes in summer and 795 vs. 540 minutes in winter) (Table S2), and they exposed larger areas of the body in the summer (median 30.0 vs. 22.6%; P = 0.0002). Both groups reduced their body exposure in winter to similar levels (12.5%), although the relative winter decrease was greater for Xhosa participants. There was limited sunscreen use in both groups (Table S2). Thus, Xhosa participants may partly compensate for their darker skin pigmentation by increasing their PNUVB in the summer (median 42,982 vs. 20,603 J; P < 0.0001), which is reflected in higher 25(OH)D levels in summer in Xhosa partic- ipants than in Cape Mixed participants (Table 1). However, Xhosa participants did not maintain higher UVB exposure in the winter (PNUVB 1,304 J vs. 1,450 J; P = 0.63), and they were at greater risk of deficiency, with 18% of participants exhibiting severe deficiency.

Genetic Variation Has a Larger Effect on the Response to Supplementation than on Seasonal Deficiency. To investigate the effects of genetic variation on serum 25(OH)D concentrations, 10 SNPs in six genes associated with vitamin D deficiency were genotyped in all par- ticipants: DBP (rs7041 and rs4588), DHCR7 (7-DCH reductase

A B C

D E F

G H I

Fig. 1. Vitamin D status, dietary vitamin D intake, and personal UVB exposure of Xhosa and Cape Mixed participants in Cape Town, South Africa, in summer, winter, and after receiving vitamin D

in winter.

(A) Study design. (B and C) Serum 25(OH)D concen- tration stratified by season (B) and after receiving vi- tamin D

(C). Dotted lines indicate status thresholds:

insufficiency, <75 nmol/L; deficiency, <50 nmol/L; severe deficiency, <30 nmol/L. (D and E) Serum 25(OH)D concentration stratified by sex (female, ♀; male, ♂) in Xhosa (D) and Cape Mixed (E) participants. (F) Dietary vitamin D intake measured by the food frequency questionnaire. Dotted lines indicate EAR. (G and H) Di- etary vitamin D intake stratified by sex in Xhosa (G) and Cape Mixed (H) participants. (I) PNUVB.

Xhosa: summer, n = 50; winter n = 33; winter + vitamin D, n = 30; Cape Mixed: summer and winter, n = 50.

Medians are indicated by red lines. Significance was tested by the Wilcoxon rank test between seasons, by the Friedman test with Dunn ’s multiple com- parisons test for 25(OH)D postsupplementation (n = 30), and by the Mann –Whitney test between pop- ulations and sex; *P < 0.05; **P < 0.01; ***P < 0.001;

****P < 0.0001.

and rs12785878), CYP2R1 (vitamin D 25-hydroxylase and rs10741657), CYP24A1 (vitamin D 24-hydroxylase and rs6013897), CYP27B1 (vitamin D 1α-hydroxylase and rs10877012), and VDR (rs2544037, rs10783219, rs10735810, and rs731236) (9–11, 20). All were in Hardy–Weinberg equilibrium, and five SNPs (CYP27B1 rs10877012; VDR rs10783219 and rs731236 TaqI; and DBP rs7041 and rs4588) had significantly higher minor allele frequency (MAF) in the Cape Mixed participants (P < 0.03, Table 2). DBP rs7041 and rs4588 are combined to form the Group component (Gc) haplotype of which there are three major alleles, Gc1F, Gc1S, and Gc2, with the Gc1F protein having higher binding affinity for serum 25(OH)D (8). Xhosa participants had significantly higher frequency of Gc1F/Gc1F carriers (76 vs. 34%), whereas the most common haplotype combination in the Cape Mixed participants

was Gc1F/Gc1S (38 vs. 18%; P = 0.0002) (Table 2). Serum DBP levels also were measured in all participants; there was no effect of season, vitamin D supplementation or Gc haplotype on serum DBP levels in either population, but Cape Mixed participants had lower median levels in both seasons (summer/winter:106/107 vs. 125/127 μg/mL; P ≤ 0.0092) (Fig. S2 E and F).

Although the powering was modest, genotypes were added to the stepwise regression and general linear models (GLM) for determinants of total 25(OH)D (Table 3 and Tables S3 and S4).

In exploratory analyses of combined participants, a greater ge- notypic effect was observed after supplementation than with seasonal variation; those heterozygous for VDR FokI (rs10735810) had lower 25(OH)D in the winter and postsupplementation (P <

0.04); those heterozygous for CYP24A1 rs6013897 had lower Table 1. Serum 25(OH)D, dietary vitamin D intake, and UVB exposure by season and population

Measure Summer Winter P value*

Serum 25(OH)D, median nmol/L (range): Xhosa 72.6 (62.1 –80.43)

45.4 (35.7 –51.2) <0.0001 Serum 25(OH)D, median nmol/L (range): Cape Mixed 65.5 (54.6–76.1) 43.8 (33.5–54.2) <0.0001

Vitamin D status, nmol/L (%) <0.0001

Xhosa: Severe deficiency, <30 nmol/L 0 6 (18)

Xhosa: Deficiency, <50 nmol/L 2 (4) 21 (64)

Xhosa: Insufficiency, 50 –75 nmol/L 28 (56) 11 (33)

Xhosa: Sufficiency, >75 nmol/L 20 (40) 1 (3)

Cape Mixed: Severe deficiency, <30 nmol/L 0 6 (12)

Cape Mixed: Deficiency, <50 nmol/L 8 (16) 35 (70)

Cape Mixed: Insufficiency, 50 –75 nmol/L 27 (54) 15 (30)

Cape Mixed: Sufficiency, >75 nmol/L 15 (30) 0

Dietary vitamin D intake, median IU/d (IQR): Xhosa 213 (94 –335) 170 (68 –266) 0.26

Dietary vitamin D intake, median IU/d (IQR): Cape Mixed 235 (120 –395) 230 (99 –369) 0.24 Personal weekly UVB, median J (IQR): Xhosa 42,982 (28,651 –90,552)

1,304 (875.5 –2,878) <0.0001 Personal weekly UVB, median J (IQR): Cape Mixed 20,603 (10,887 –42,897) 1,450 (392.6 –3,530) <0.0001

*Wilcoxon rank test or Fisher ’s exact test between seasons, bold type indicates statistically significant difference between summer and winter values.

†

Mann –Whitney test significant between populations, P < 0.04, bold type indicates statistically significant difference between summer and winter values.

Table 2. SNP frequency in Xhosa and Cape Mixed participants

Gene and SNP (also known as) Location (function) Allele* Xh (Major) Xh (Het) Xh (Minor) CM (Major) CM (Het) CM (Minor) P-value

CYP2R1 rs10741657 (rs2060793)

Promoter A/G 0.72 0.22 0.06 0.54 0.38 0.08 0.17

CYP27B1 rs10877012 (rs4646536)

5 ′ UTR C/A 0.86 0.12 0.02 0.54 0.42 0.04 0.002

CYP24A1 rs6013897 3 ′ downstream A/T 0.52 0.42 0.06 0.66 0.26 0.08 0.24

DBP

rs7041 (Asp416Glu) Exon 11 (nonsyn) C/A

0.8 0.18 0.02 0.5 0.44 0.06 0.007

rs4588 (Thr420Lys) Exon 11 (nonsyn) T/G

0.96 0.04 0 0.78 0.2 0.02 0.027

VDR

rs2544037 Promoter A/G 0.52 0.38 0.1 0.58 0.3 0.12 0.7

rs10783219 Intron 0 A/T 0.9 0.1 0 0.72 0.22 0.06 0.044

rs10735810 (FokI) Exon 3 (nonsyn) A/G 0.64 0.3 0.06 0.56 0.38 0.06 0.69

rs731236 (TaqI) Intron 9 A/G 0.72 0.28 0 0.54 0.4 0.06 0.069

DHCR7 rs12785878 (rs7944926;

rs3794060)

Intron 2 G/T

0.52 0.42 0.06 0.5 0.44 0.06 0.98

DBP Gc Haplotype (alleles rs7041 –rs4588)

Gc1F/Gc1F Exon 11 (nonsyn) CC-TT 0.76 0.34

Gc1F/Gc2 Exon 11 (nonsyn) CC-TG 0.04 0.14

Gc2/Gc2 Exon 11 (nonsyn) CC-GG 0 0.02

Gc1F/Gc1S Exon 11 (nonsyn) CA-TT 0.18 0.38

Gc2/Gc1S Exon 11 (nonsyn) CA-GT 0 0.06

Gc1S/Gc1S Exon 11 (nonsyn) AA-TT 0.02 0.06

CM, Cape Mixed; Het, heterozygous; Xh, Xhosa.

*Major/minor alleles and frequencies.

†

P values for differences between sites tested by χ

test for trend, bold type indicates statistically significant difference in SNP frequencies.

‡

In linkage disequilibrium, r

= 1.00 (9, 10).

§

On reverse strand.

{

Predicted according to ref. 21.

MEDICALSCIENCESEARTH,ATMOSPHERIC, ANDPLANETARYSCIENCES

25(OH)D levels postsupplementation (P = 0.006); and those het- erozygous for CYP2R1 rs10741657 and DBP rs7041 had higher 25(OH)D levels postsupplementation (P = 0.002) (Fig. S3).

Personal UVB Exposure Is the Major Determinant of Vitamin D Status.

We next used regression models applied to all variables (SI Materials and Methods) to identify the determinants of serum 25(OH)D in both groups. Stepwise regression (Table S3) iden- tified PNUVB as the dominant determinant (F = 123.2, P <

0.0001), followed by area of skin exposed (F = 10.7, P = 0.0013) and arm MI (F = 7.40, P = 0.0072). DBP haplotype Gc1F/Gc1S and two VDR SNPs, TaqI rs731236 and rs2544037, also con- tributed to the model, as did duration of UVB exposure and skin redness (arm EI), to a lesser extent. To determine the directionality of effect a GLM approach was applied adjusting for age and smoking status (Table 3). Again PNUVB was the dominant determinant, followed by area of skin exposed and weekly duration of exposure, all positively contributing to serum 25(OH)D, whereas VDR FokI rs10735810 AG and Cape Mixed ancestry where negatively correlated with 25(OH)D. The two measures of skin redness (arm and forehead EI), as well as Gc1F/Gc1S, VDR FokI rs10735810 AA, and DBP rs7041 CA, positively contributed to serum 25(OH)D.

The determinants of severe deficiency in winter and sufficiency in the summer also were examined using a GLM approach (Table S4). Sunlight exposure as a component of PNUVB did not prevent serious deficiency in the winter, but the area exposed and duration were important. The UVB content of winter sunlight is weak, but individuals who were in the sun for longer periods and who had the most surface area exposed produced more vitamin D. Darker skin (higher arm and forehead MI) also was associated with lower serum 25(OH)D status. Sex had an effect but corre- lated with area exposed and duration of exposure. Sufficiency in the summer was affected most strongly by duration of exposure and PNUVB and to a lesser extent by the degree of skin redness (arm EI). Possession of the DHCR7 rs12785878 GG genotype had a small effect on optimal serum 25(OH)D, but no other genes had a significant effect. Smoking status and sex also were influential in summer but only through correlation with patterns of sun exposure and skin pigmentation, respectively.

Winter Vitamin D

Supplementation Increases Peripheral WBC Count and Counteracts Anemia. To investigate the functional consequence of seasonal serum 25(OH)D levels on the immune system, we next investigated seasonal differences in full blood count (FBC) and the effect of vitamin D

supplementation on FBC in Xhosa participants.

Vitamin D

supplementation in the winter increased WBC count (P = 0.0016) and in particular lymphocyte count (P = 0.023), and there was a winter trend for decreased monocytes (Fig. 2 A–C).

In the winter, participant’s RBC parameters tended toward mac- rocytic anemia [evidenced by decreased RBC and RBC dis- tribution width (RDW), increased mean corpuscular volume, and

a trend toward decreased Hb], and this tendency was reversed by supplementation (P ≤ 0.0007) (Fig. 2 D–F and Fig. S4A). The winter decline in RBC, RDW, and Hb also was seen in participants with Cape Mixed ancestry, although the effect of supplementation was not measured (Fig. S4 B–D).

Winter Vitamin D

Supplementation Decreases HIV-1 Replication in Peripheral Blood Mononuclear Cells. Because supplementation modified peripheral WBC and RBC counts, we next investigated the functional consequences on response to HIV-1 infection.

The active vitamin D metabolite, 1α,25-dihydroxy-vitamin D, has been shown in vitro to inhibit HIV-1 replication in macrophages via induction of autophagy, mediated via cathelicidin induction (22), and 25(OH)D deficiency is associated with HIV-1 pro- gression (1). Therefore we investigated the functional conse- quences of seasonal variation in serum 25(OH)D levels and winter vitamin D

supplementation on the extent of HIV-1 replication in freshly isolated peripheral blood mononuclear cells (PBMCs), at each study visit. PBMCs were cultured in the presence of fresh 20% autologous serum, isolated at the same time as PBMCs, to maintain the in vivo cellular environment as best possible, with regard to seasonal serum 25(OH)D levels, autologous DBP, and other circulating chemokines/cytokines.

Two preparations of HIV-1 BaL, purified and unpurified, were used for infection. Unpurified HIV-1 preparations contain exo- somes, microvesicles, and conditioned medium from propagation and were tested for the likelihood that activating cells might result in greater productive infection of unstimulated cells.

Infection of PBMCs in winter, compared with summer, re- sulted in greater productive HIV-1 infection on day 9, as mea- sured by culture supernatant p24 antigen levels. This result was seen in PBMCs isolated from both Xhosa (P = 0.0003, Fig. 2 G–I) and Cape Mixed (P < 0.0001, Fig. S4 E and F) participants.

This winter increase in HIV-1 infection was seen with both preparations of HIV-1, with ∼1-log higher p24 measured from cells infected with unpurified virus (Xhosa median 7,038 pg/mL unpurified vs. 738 pg/mL purified) (Fig. 2 H and I).

After 6 wk of vitamin D

supplementation in winter, the winter increase in HIV-1 p24 was attenuated, and Xhosa participants’

PBMCs showed a diminished capacity for productive HIV-1 in- fection: on day 9 p24 levels had dropped to the level as observed in summer (Fig. 2G). Again, this decrease occurred with in- fections using both purified and unpurified virus (Fig. 2 H and I), demonstrating the robustness of oral vitamin D

supplementa- tion in suppressing productive HIV-1 infection in peripheral blood cells ex vivo. Moreover there was a significant negative correlation between paired serum 25(OH)D and day 9 p24 concentrations from PBMCs infected with purified virus, across all time points (Spearman r

= −0.36; P < 0.0001), indicating a direct correlation between serum 25(OH)D levels and the ability of peripheral blood cells to limit productive HIV-1 infection.

Discussion

The multitude of, and complex interactions among, variables that modify vitamin D status and their impact on immunological function are poorly understood, particularly in the context of disease prevention in healthy individuals. In an urban African location with seasonal UVB variability and high infectious dis- ease prevalence, we found that personal UVB exposure habit is the most important determinant of vitamin D status in healthy adults with moderate to dark skin pigmentation. Moreover, we found that the reversal of vitamin D deficiency in winter through oral supplementation can modulate the number and function of circulating WBCs, prevent anemia, and decrease productive HIV-1 infection. Furthermore, we found that UVB exposure habit can counterbalance the effects of pigmentation, genetic variation, and poor dietary intake.

The skin of the indigenous people of the Cape, the Khoisan, is considerably lighter than that of either study group (23) and may represent a long-established adaptation to seasonal UVB. The darker skin of both study populations denotes a degree of Table 3. Determinants of serum 25(OH)D concentration

Variable t-statistic R-statistic P value*

PNUVB 9.502 0.579 <0.0001

Area of skin exposed 5.855 0.401 <0.0001 Weekly duration of UVB 4.401 0.312 <0.0001

Arm EI 3.617 0.261 0.0004

Gc1F/Gc1S 3.026 0.221 0.0028

VDR rs10735810 AA 2.715 0.199 0.0073

DBP rs7041 CA 2.656 0.195 0.0086

Forehead EI 2.368 0.174 0.0190

Cape Mixed ancestry −2.271 −0.167 0.0243

VDR rs10735810 AG −2.544 −0.187 0.0118

*P values derived using linear regression, with adjustment for covariates

(smoking and age). The false-discovery rate was determined by the Benja-

mini –Hochberg; all <0.1.

mismatch between skin pigmentation and environmental UVB resulting from their recent migration into the region; this effect is exacerbated by wearing concealing clothing and indoor living in the winter. The high prevalence of vitamin D deficiency in the winter in both groups indicates that people with moderate or dark pigmentation are at high risk of deficiency in the absence of significant dietary vitamin D intake when UVB radiation is limited by seasonal fluctuations.

We also noted significant polymorphic variation between the two populations for 5 of the 10 vitamin D-associated SNPs in- vestigated, contributing further genetic insight into these under- studied populations. Given the low MAF of the SNPs investigated, particularly in the Xhosa population, we identified only a minor effect of genetic variation on seasonal vitamin D status. This finding mirrors the recent genomewide association study in 16,125 individuals, from five cohorts, which showed the pro- portion of variation in 25(OH)D attributable to genetic variation was 1–4% (9). However, despite the small sample size, we identified four SNPs, in VDR, CYP24A1, CYP2R1, and DBP, which modify the response to high-dose supplementation. Al- though these results need to be confirmed in larger cohorts, such associations are supported by our previous finding that polymorphic variation in VDR modifies the effect of high-dose vitamin D sup- plementation on hastening time to sputum culture conversion in TB patients receiving intensive-phase treatment (24).

In South Africa, young females are at the greatest risk of HIV-1 infection (19). We found females in both populations also were at greater risk of deficiency, indicating this is an important target group for intervention. Dietary intake played a greater role in maintaining serum 25(OH)D levels when UVB levels were low.

However, it is unlikely that a diet richer in vitamin D-containing foods alone can compensate for the seasonal changes in in- solation and UVB at this or higher latitudes, especially when colder outdoor temperatures and the associated wearing of clothes and indoor living reduce the likelihood of exposure to weak UVB-containing sunlight. Recommendations for personal vitamin D supplementation or wide-scale food fortification may be considered, along with season-specific recommendations for short-duration sun exposure around noon in the winter and be- tween ∼1.0 and 1.5 h sun exposure in the spring and autumn.

Short periods of UVB exposure around solar noon are highly effective in raising serum 25(OH)D levels and pose a low risk to general and skin health (25).

Our findings of decreased winter WBC counts, particularly lymphocytes, and an increase in numbers following vitamin D supplementation corroborate a small longitudinal study in healthy Scandinavian adults with light pigmentation, which found a similar decrease in lymphocytes in winter, particularly CD4

and CD8

T cells, which was associated with reduced 25(OH)D (26). Higher serum 25(OH)D levels in children initiating antiretroviral therapy (ART) also were associated with higher CD4

T-cell restoration (27). Further studies will investigate the detailed changes in in- nate and adaptive immune cell populations in our cohorts. Vi- tamin D deficiency also has been associated previously with anemia and low Hb in HIV-infected women (1). We found that vitamin D supplementation reversed winter-associated macro- cytic anemia, suggesting that this adjunct therapy also may be effective in preventing anemia in HIV-infected individuals.

The demonstration that high-dosage oral vitamin D supple- mentation reversed serum 25(OH)D deficiency and attenuated the seasonal increase in ex vivo HIV-1 replication, similar to our previous finding that oral vitamin D reduces Mycobacterium tuber- culosis replication in whole blood (28), provides strong evidence for the positive preventative effects of vitamin D supplementation for people with vitamin D deficiency and serious infectious diseases, conditions which apply to many cities in which the prevalence of vitamin D deficiency continues to rise. Furthermore, vitamin D may be a simple, cost-effective intervention, particularly in re- source-poor settings, to prevent disease progression in persons infected with HIV-1 by suppressing viral replication, raising pe- ripheral lymphocyte counts, and preventing anemia, potentially prolonging the time to ART initiation and enhancing the bene- ficial effects of ART once initiated.

Materials and Methods

Study Design. The study was conducted in accordance with the Helsinki 1964 declaration, including subsequent revisions, and the South African Guidelines for Good Clinical Practice and the Medical Research Council Ethical Guidelines for Research. Ethical approval was received from the Human Research Ethics Review Boards of the Faculty of Health Sciences, University of Cape Town (ref. 003/2013) and the Faculty of Medicine and Health Sciences, Stellen- bosch University (ref. N12/10/065) and from the Institutional Review Board of The Pennsylvania State University (ref. 41940). Written informed consent was obtained from all participants.

The 104 participants were assessed for eligibility, and 100 HIV-1 – uninfected individuals (Xhosa, n = 50; Cape Mixed, n = 50) were enrolled.

The summer and winter visits began 6 wk postsolstice. At the winter visit, all

3 6 9

101 102 103 104 105

LOD

Days post-infection

HIV-1 p24 (pg/ml)

Summer Winter Winter + Vitamin D

********

Xhosa - purified HIV-1

3 6 9

101 10² 10³ 10⁴ 105 106

Days post-infection

HIV-1 p24 (pg/ml)

Summer Winter Winter + Vitamin D

****

Xhosa - unpurified HIV-1 Summer Winter Winter + VitD

10¹ 10² 10³ 10⁴ 10⁵ 10⁶ 10⁷

Day 9 HIV-1 p24 (pg/ml)LOD

*** ^ns

Xhosa - purified HIV-1

Summer Winter Winter + Vitamin D 2

3 4 5 6 7

Red Blood Cells (x1012/L)

Xhosa

**** ****

Summer Winter Winter + Vitamin D 10

15 20 25

Xhosa

*

**** ****

Summer Winter Winter + Vitamin D 2

4 6 8 10 12 14 16

White Blood Cells (x109/L)

Xhosa

* **

Summer Winter Winter + Vitamin D 0

2 4 6 8 10

Lymphocytes (x109/L)

*

Xhosa

**

A B C

D E F

G H I

Summer Winter Winter + Vitamin D 70

80 90 100

110 ***

Xhosa

Summer Winter Winter + Vitamin D 0.0

0.2 0.4 0.6 0.8 1.0 1.2

Monocytes (x109/L)

Xhosa 0.3

Fig. 2. FBC and HIV-1 replication in PBMCs from Xhosa participants in summer, winter, and after re- ceiving winter vitamin D supplementation. (A –F) Box plots show WBC (A), lymphocyte (B), monocyte (C), and RBC (D) counts, RBC distribution width (E ), and mean corpuscular volume (F) measured at each study visit (summer, n = 42; winter, n = 23; winter + vitamin D, n = 30). The lines across the box plots indicate the median (minimum –maximum); Kruskal–

Wallis with Dunn ’s multiple comparison test. (G) HIV- 1 p24 concentration in culture supernatant 9 d post- infection of PBMCs (n = 30 longitudinally; the line indicates the median) with purified HIV-1 on the day of phlebotomy in 20% autologous serum; Fried- man test with Dunn ’s multiple comparison test. (H and I) HIV-1 p24 concentration in culture supernatant on day 3, 6, and 9 postinfection of PBMCs [n = 30 lon- gitudinally, median (IQR)] with purified HIV-1 (H) or unpurified HIV-1 (I); repeated measures two-way ANOVA with Tukey ’s multiple comparison test fol- lowing log

transformation. *P < 0.05; **P < 0.01;

***P < 0.001; ****P < 0.0001. LOD, limit of detection (1 pg/mL); ns, not significant.

MEDICALSCIENCESEARTH,ATMOSPHERIC, ANDPLANETARYSCIENCES

participants received six capsules of 50,000 IU cholecalciferol (D3-50; Biotech Pharmaceutical), to be taken weekly, and Xhosa participants were followed up 6 wk ( ± 15 d; mean +3 d) after their winter visit ( Fig. S1). Previous studies have shown that serum 25(OH)D plateaus after ∼4–6 wk of supplementation (29); however, it is possible that the final equilibrated 25(OH)D level was underestimated at 6 wk. Ethics, recruitment, follow-up, and exclusion cri- teria are detailed in SI Materials and Methods. The procedures described below were conducted at each study visit.

UVB Exposure. Sun exposure was assessed at each visit by a retrospective questionnaire that captured duration and time of day of outdoor sun ex- posure and the amount of skin not covered by clothing or a hat at time of exposure. PNUVB was calculated as described in SI Materials and Methods.

Direct measurements of UVB were made at the University of Stellenbosch Solar Resource and Weather Station.

Skin Reflectometry. Skin reflectance of all research subjects, expressed as EI and MI, was measured using a portable reflectometry device (DSM II Col- orMeter; Cortex Technology). Constitutive pigmentation was measured on the upper inner arm site, and facultative pigmentation was measured on the forehead. Three independent measures were taken from each site, and the mean was calculated.

Food Questionnaire. Intake of vitamin D (vitamin D

and vitamin D

) was estimated using a 7-d quantitative food frequency questionnaire adminis- tered to every participant by a trained researcher at each study visit. The questionnaire (described in SI Materials and Methods) was adapted from a food frequency questionnaire shown to be valid in providing a reasonable estimation of dietary vitamin D intake in healthy young adults of diverse ancestry (30). Individuals taking vitamin D supplements were excluded.

Biochemistry and FBC. Peripheral blood collected in serum tubes was analyzed on the day of collection for 25(OH)D concentration by the chemiluminescent LIAISON 25 OH Vitamin D TOTAL Assay (DiaSorin). Serum also was stored at

−80 °C and subsequently batch analyzed for DBP, acute phase markers,

calcium, and albumin. FBCs were conducted within 2 –3 h of collection. As- says are described in SI Materials and Methods.

SNP Genotyping. DNA was extracted from PBMCs using the QIAmp DNA Blood Mini Kit (QIAGEN), and 10 ng was analyzed by TaqMan Genotyping assay (Life Technologies), in duplicate, for the following SNPs in genes: DBP (rs7041 and rs4588), DHCR7 (rs12785878), CYP2R1 (rs10741657), CYP24A1 (rs6013897), CYP27B1 (rs10877012), and VDR (rs2544037, rs10783219, rs10735810, and rs731236), as described in SI Materials and Methods.

PBMC HIV-1 Infection and p24 Analysis. After the isolation of PBMCs, cells were infected immediately with HIV-1, as described in SI Materials and Methods.

HIV-1 replication was measured by quantifying the HIV-1 p24 antigen con- centration of lysed supernatant by Luminex, as described in ref. 31, with slight modifications (SI Materials and Methods).

Statistical Analysis. All univariate statistics were conducted in GraphPad Prism 6.0 software with an alpha of 0.05 and two-sided testing. GLM was conducted using Qlucore Omics Explorer 2.2 software and stepwise regression, and GLM on vitamin D optimality and deficiency was conducted in S-Plus. Full details are given in SI Materials and Methods.

ACKNOWLEDGMENTS. We thank all participants for their voluntary partic- ipation; C. Diedrich, B. Alexander, and E. Geldenhuys for research assistance;

and A. Martineau and D. Jolliffe for assistance with SNP selection. UVB data were collected at the Solar Technology and Energy Research Group solar observatory of Stellenbosch University, with assistance from P. Gauché and M. Mouzouris. This work was supported by a John Simon Guggenheim Fel- lowship; the Stellenbosch Institute for Advanced Study (N.G.J. and G.C.); a Sydney Brenner Postdoctoral Fellowship, Academy of Science of South Africa (A.K.C.); South African Sugar Association Research Grant 232 (to C.E.N.); UK Medical Research Council Grant U1175.02.002.00014; European Union 7

Framework Programme for Research and Technological Development Grant Health-F3-2012-305578; and Wellcome Trust Grants 084323 and 088316 (to R.J.W.).

1. Mehta S, et al. (2010) Vitamin D status of HIV-infected women and its association with HIV disease progression, anemia, and mortality. PLoS ONE 5(1):e8770.

2. Sudfeld CR, et al. (2012) Vitamin D and HIV progression among Tanzanian adults initiating antiretroviral therapy. PLoS ONE 7(6):e40036.

3. Holick MF, et al. (1980) Photosynthesis of previtamin D3 in human skin and the physiologic consequences. Science 210(4466):203–205.

4. Holick MF, Chen TC, Lu Z, Sauter E (2007) Vitamin D and skin physiology: A D-lightful story. J Bone Miner Res 22(Suppl 2):V28–V33.

5. Webb AR (2006) Who, what, where and when-influences on cutaneous vitamin D synthesis. Prog Biophys Mol Biol 92(1):17–25.

6. Thieden E, Philipsen PA, Heydenreich J, Wulf HC (2009) Vitamin D level in summer and winter related to measured UVR exposure and behavior. Photochem Photobiol 85(6):

1480–1484.

7. Wortsman J, Matsuoka LY, Chen TC, Lu Z, Holick MF (2000) Decreased bioavailability of vitamin D in obesity. Am J Clin Nutr 72(3):690–693.

8. Arnaud J, Constans J (1993) Affinity differences for vitamin D metabolites associated with the genetic isoforms of the human serum carrier protein (DBP). Hum Genet 92(2):183–188.

9. Wang TJ, et al. (2010) Common genetic determinants of vitamin D insufficiency: A genome-wide association study. Lancet 376(9736):180–188.

10. Engelman CD, et al. (2008) Genetic and environmental determinants of 25-hydrox- yvitamin D and 1,25-dihydroxyvitamin D levels in Hispanic and African Americans.

J Clin Endocrinol Metab 93(9):3381–3388.

11. Ramos-Lopez E, et al. (2008) Gestational diabetes mellitus and vitamin D deficiency:

Genetic contribution of CYP27B1 and CYP2R1 polymorphisms. Diabetes Obes Metab 10(8):683–685.

12. Uitterlinden AG, Fang Y, Van Meurs JB, Pols HA, Van Leeuwen JP (2004) Genetics and biology of vitamin D receptor polymorphisms. Gene 338(2):143–156.

13. Holick MF, Matsuoka LY, Wortsman J (1989) Age, vitamin D, and solar ultraviolet.

Lancet 2(8671):1104–1105.

14. Liu PT, et al. (2006) Toll-like receptor triggering of a vitamin D-mediated human antimicrobial response. Science 311(5768):1770–1773.

15. Pinzone MR, et al. (2013) LPS and HIV gp120 modulate monocyte/macrophage CYP27B1 and CYP24A1 expression leading to vitamin D consumption and hypovitaminosis D in HIV-infected individuals. Eur Rev Med Pharmacol Sci 17(14):1938–1950.

16. Ross AC (2011) The 2011 report on dietary reference intakes for calcium and vitamin D. Public Health Nutr 14(5):938–939.

17. Brot C, Jorgensen NR, Sorensen OH (1999) The influence of smoking on vitamin D status and calcium metabolism. Eur J Clin Nutr 53(12):920–926.

18. Patterson N, et al. (2010) Genetic structure of a unique admixed population: Impli- cations for medical research. Hum Mol Genet 19(3):411–419.

19. Abdool Karim Q, Abdool Karim SS, Singh B, Short R, Ngxongo S (1992) Seroprevalence of HIV infection in rural South Africa. AIDS 6(12):1535–1539.

20. Uitterlinden AG, Fang Y, van Meurs JB, van Leeuwen H, Pols HA (2004) Vitamin D receptor gene polymorphisms in relation to Vitamin D related disease states. J Steroid Biochem Mol Biol 89-90(1-5):187–193.

21. Martineau AR, et al. (2010) Association between Gc genotype and susceptibility to TB is dependent on vitamin D status. Eur Respir J 35(5):1106–1112.

22. Campbell GR, Spector SA (2011) Hormonally active vitamin D3 (1alpha,25-dihydrox- ycholecalciferol) triggers autophagy in human macrophages that inhibits HIV-1 in- fection. J Biol Chem 286(21):18890–18902.

23. Weiner JS, Harrison GA, Singer R, Harris R, Jopp W (1964) Skin colour in southern Africa. Hum Biol 36(3):294–307.

24. Coussens AK, et al. (2012) Vitamin D accelerates resolution of inflammatory responses during tuberculosis treatment. Proc Natl Acad Sci USA 109(38):15449–15454.

25. Holick MF (2004) Vitamin D: Importance in the prevention of cancers, type 1 diabetes, heart disease, and osteoporosis. Am J Clin Nutr 79(3):362–371.

26. Khoo A-L, et al. (2012) Seasonal variation in vitamin D3levels is paralleled by changes in the peripheral blood human T cell compartment. PLoS ONE 7(1):e29250.

27. Ross AC, et al. (2011) Vitamin D is linked to carotid intima-media thickness and im- mune reconstitution in HIV-positive individuals. Antivir Ther 16(4):555–563.

28. Martineau AR, et al. (2007) A single dose of vitamin D enhances immunity to myco- bacteria. Am J Respir Crit Care Med 176(2):208–213.

29. Vieth R (1999) Vitamin D supplementation, 25-hydroxyvitamin D concentrations, and safety. Am J Clin Nutr 69(5):842–856.

30. Wu H, et al. (2009) The development and evaluation of a food frequency question- naire used in assessing vitamin D intake in a sample of healthy young Canadian adults of diverse ancestry. Nutr Res 29(4):255–261.

31. Biancotto A, et al. (2009) A highly sensitive and dynamic immunofluorescent cyto- metric bead assay for the detection of HIV-1 p24. J Virol Methods 157(1):98–101.

32. Langenhoven ML, Conradie PJ, Wolmarans P, Faber M (1991) MRC Food Quantities Manual (South African Medical Research Council, Cape Town, South Africa).

33. Lange CM, et al. (2012) A genetic validation study reveals a role of vitamin D me- tabolism in the response to interferon-alfa-based therapy of chronic hepatitis C. PLoS ONE 7(7):e40159.

34. Noursadeghi M, et al. (2009) Genome-wide innate immune responses in HIV-1- infected macrophages are preserved despite attenuation of the NF-kappa B ac- tivation pathway. J Immunol 182(1):319–328.

35. Wei X, et al. (2002) Emergence of resistant human immunodeficiency virus type 1 in patients receiving fusion inhibitor (T-20) monotherapy. Antimicrob Agents Chemo- ther 46(6):1896–1905.

36. Troyanskaya O, et al. (2001) Missing value estimation methods for DNA microarrays.

Bioinformatics 17(6):520–525.

37. Wichura MJ (2006) The Coordinate-Free Approach to Linear Models (Cambridge Univ.

Press, Cambridge, UK).